Ang (1-7) derviative oligopeptides for the treatment of pain

ABSTRACT

The present invention provides oligopeptides, in particular, Ang-(1-7) derivatives, and methods for using and producing the same. In one particular embodiment, oligopeptides of the invention have higher blood-brain barrier penetration and/or in vivo half-life compared to the native Ang-(1-7), thereby allowing oligopeptides of the invention to be used in a wide variety of clinical applications including in treatment of cognitive dysfunction and pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/109,604, filed Dec. 2, 2020, which is a continuation of U.S.application Ser. No. 16/441,767, filed Jun. 14, 2019, which is acontinuation of International Patent Application No. PCT/US18/12893,filed Jan. 9, 2018, which is a continuation of U.S. application Ser. No.15/401,944, filed Jan. 9, 2017, now U.S. Pat. No. 10,183,055, which is acontinuation-in-part of U.S. application Ser. No. 15/134,073, filed Apr.20, 2016, now U.S. Pat. No. 9,670,251, which is a continuation of U.S.application Ser. No. 14/801,557, filed Jul. 16, 2015, now U.S. Pat. No.9,796,759, which claims the priority benefit of and priority to U.S.Provisional Application No. 62/027,219, all of which are incorporatedherein by reference in their entireties.

SEQUENCE LISTING

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 31,000 Byte ASCII (Text) file named“39188-308_SequenceListing” created on Jan. 9, 2023.

FIELD OF THE INVENTION

The present invention relates to oligopeptides, such as Ang-(1-7) andrelated derivative oligopeptides, and methods for using the same for thetreatment of pain of various etiologies.

BACKGROUND OF THE INVENTION

Bone pain is experienced by 75-90% of late-stage metastatic cancerpatients. Metastatic cancer-induced bone pain (CIBP) is frequentlyreported but poorly managed. The World Health Organization implemented athree-tiered Pain-Relief-Ladder for cancer pain that recommendsmild-to-strong opioids as the cancer progresses. Yet, moderate-to-severecancer pain is not adequately managed in many patients with currentanalgesic therapy. Opioid therapy is associated with a host ofchallenging side effects contributing to their failure, while diversionof prescribed opioids has led to an addiction epidemic. Recent reportssuggest that opioids may exacerbate bone loss in humans and experimentalanimal models, indicating that opioid therapy may be counterproductiveto anti-osteolytic co-therapies and CIBP management. Furthermore,prolonged opioid therapy may increase the proliferation/migration ofcertain cancers.

Preclinical modeling of CIBP has revealed mechanisms driving thiscomplex disease state and lead to the identification of potentialtherapeutic targets. Although the bone is innervated by both sympatheticand nociceptive nerve fibers, many human tumors of the bone lackdetectable nerve fibers in the tumor itself and adjacent peripheralbone. Contributors to nociceptive signaling associated with CIBP includean acidic tumor environment and the secretion of growth factors,cytokines, and chemokines from the tumor and tumor-associated cells, aswell as enhanced nerve sprouting in the local environment.

The bone is innervated by both sympathetic and nociceptive nerve fibers.However, many human bone tumors lack detectable nerve fibers within thetumor itself and the adjacent peripheral bone. Contributors tonociceptive signaling associated with CIBP include an acidic tumorenvironment and the secretion of growth factors, cytokines, andchemokines from the tumor and tumor-associated cells, as well asenhanced nerve sprouting in the local environment. Thus, there is a needto develop non-opioid analgesics for the treatment of pain includingcancer-induced bone pain.

The present inventions are based on the discovery that native Ang(1-7),related derivative polypeptides, and/or non-peptide agonists that haveaffinity and agonistic efficacy for the Mas receptor improve a varietyof biologic, physiologic, and pathologic parameters. Specifically, it isshown that Mas receptor activation attenuates spatial memory and objectrecognition impairment caused by congestive heart failure (CHF), pain ofvarious etiologies including cancer-induced bone pain and theneurological sequelae of traumatic brain injury (TBI).

SUMMARY OF THE INVENTION

Some aspects of the invention provide an oligopeptide that is anon-naturally-occurring angiotensin-(1-7) derivative polypeptide, i.e.,“Ang-(1-7) derivative.” Oligopeptides of the invention may have a longerin vivo half-life and/or increased blood-brain barrier penetration thanAng-(1-7). In some embodiments, the oligopeptides of the invention haveseven or eight amino acids and have biological activity as an agonist ofthe Mas receptor.

One particular aspect of the invention provides an oligopeptidederivative of the formula: A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ ID NO:1), whereA¹ is selected from the group consisting of aspartic acid, glutamicacid, alanine, and glycosylated forms thereof; A² is selected from thegroup consisting of arginine, histidine, lysine, and glycosylated formsthereof; A³ is selected from the group consisting of valine, alanine,isoleucine, leucine, and glycosylated forms thereof; A⁴ is selected fromthe group consisting of tyrosine, phenylalanine, tryptophan, andglycosylated forms thereof; A⁵ is selected from the group consisting ofisoleucine, valine, alanine, leucine, and glycosylated forms thereof; A⁶is selected from the group consisting of histidine, arginine, lysine,and glycosylated forms thereof; A⁷ is selected from the group consistingof proline, glycine, serine, and glycosylated forms thereof; and A⁸ canbe present or absent, wherein when A⁸ is present, A⁸ is selected fromthe group consisting of serine, threonine, hydroxyproline, andglycosylated forms thereof, provided (i) at least one of A¹-A⁸ isoptionally substituted with a mono- or di-carbohydrate; or (ii) when A⁸is absent: (a) at least one of A¹-A⁷ is substituted with a mono- ordi-carbohydrate, (b) A⁷ is terminated with an amino group, or (c) acombination thereof.

In some embodiments, carbohydrate comprises glucose, galactose, xylose,fucose, rhamnose, lactose, cellobiose, melibiose, or a combinationthereof. In other embodiments, A⁸ is serine or a glycosylated formthereof, or A⁸ is absent and A⁷ is serine or a glycosylated formthereof. In some embodiments, only the C-terminal amino acid isglycosylated (e.g., A⁸ or A⁷ when A⁸ is absent).

Still in other embodiments, (i) A⁸ is terminated with an amino group; or(ii) when A⁸ is absent, A⁷ is terminated with an amino group. Withinthese embodiments, in some instances (i) A⁸ is serine that is optionallyglycosylated (e.g., with glucose or lactose); or (ii) when A⁸ is absent,A⁷ is serine that is optionally glycosylated (e.g., with glucose orlactose). Still in other instances, when A⁸ is absent and A⁷ serine thatis glycosylated with glucose. Within the latter instances, in some casesA⁷ is terminated with an amino group. In some embodiments, whether ornot the Ang(1-7) derivative is terminated with an amino group, theC-terminal amino acid (A⁸ or A⁷ when A⁸ is absent) is the onlyglycosylated amino acid.

Yet in other embodiments, A¹ is aspartic acid; A² is arginine; A³ isvaline; A⁴ is tyrosine; A⁵ is isoleucine; A⁶ is histidine; and (i) A⁸ isabsent and A⁷ is terminated with an amino group or A⁷ is a glycosylatedserine, or (ii) A⁸ is serine terminated with an amino group. Withinthese embodiments, in some cases A⁸ is a glycosylated serine. Still inother cases, A⁸ is absent and A⁷ is a glycosylated serine that isterminated with an amino group.

Another aspect of the invention provides a glycosylated Ang-(1-7)derivative having eight amino acids or less, typically seven or eightamino acids (e.g., amino acid residues). In some embodiments, theglycosylated Ang-(1-7) derivative is glycosylated with xylose, fucose,rhamnose, glucose, lactose, cellobiose, melibiose, or a combinationthereof. Still in other embodiments, the carboxylic acid end of saidglycosylated Ang-(1-7) derivative is substituted with an amino group.

Other aspects of the invention provide methods for treating cognitivedysfunction and/or impairment in a subject by administering atherapeutically effective amount of an oligonucleotide of the invention.In general, oligopeptides of the invention can be used to treat anyclinical condition that can be treated with Ang-(1-7).

In some embodiments, the oligopeptides of the invention may be used totreat (i.e., reduce or eliminate) pain of any etiology (i.e., a painfulcondition). Specific pain syndromes and painful conditions amenable totreatment include, for example, acute pain (e.g., trauma-induced pain),dental pain (e.g., following a tooth extraction or other dentalprocedure), cancer-induced bone pain from caused by either a primary ormetastatic tumor, post-surgical pain, post-herpatic neuralgia,fibromyalgia, inflammatory pain, stroke-induced pain, trauma-basedneuropathic pain, multiple sclerosis-induced pain, rheumatoid arthritis,osteoarthritis, and complex regional pain syndrome (CRPS). Theoligopeptides of the invention also may be used to treat pain and othersymptoms and conditions associated with HIV-induced neuropathy, diabeticneuropathy, and chemotherapeutic neuropathy.

In other embodiments, the oligopeptides of the invention may be used toreduce or eliminate one or more symptoms of cognitive impairmentgenerally, and conditions caused by or associated with vascularcontributions to cognitive impairment and dementia (“VCID”) including,for example, reduced attention, memory loss, psychomotor slowing, anddiminished executive function. Specific conditions that are associatedwith cognitive impairment and/or VCID, and that are amenable totreatment using the inventive oligopeptides include, for example,cognitive impairment caused by or associated with congestive heartfailure, cardiovascular disease, hypertension, stroke, post-operativecognitive defects and/or delerium, dementia including age-relateddementia, vascular dementia, Alzheimer's disease, and traumatic braininjury including concussion and penetrating brain injury.

In some embodiments, the inventive oligopeptides are administered at adosage of about 0.1-50 mg/kg, including for example at least about 0.25,0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10, 15, 20, 25, 30, or 40mg/kg. The oligopeptides may be administered QD, bid, tid, qid, or moreas necessary to obtain the desired clinical outcome. The oligopeptidesmay be administered orally or by injection (intravenous, subcutaneous,intramuscular, intraperitoneal, intracerebroventricular, orintrathecal), or by inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing some of the oligopeptides of the invention andnative Ang-(1-7) to activate human umbilical vascular endothelial cells(HUVEC) in culture.

FIG. 2 is a graph showing NO production assay results for nativeAng-(1-7) and oligopeptides PN-A3, PN-A4 and PN-A5 of the invention.

FIG. 3A is a graph showing the select Mas receptor antagonists A779blocks NO production induced by oligopeptide PN-A5 of the invention.

FIG. 3B is a graph showing the averaged effect of the select Masreceptor antagonists A779 on NO production induced by oligopeptidePN-A5.

FIG. 4 is a graph showing the effects of oligopeptide PN-A5 on heartfailure induced object recognition memory impairment.

FIG. 5 is a graph showing the effects of oligopeptide PN-A5 on heartfailure induced spatial memory impairment.

FIG. 6A is a graph showing oligopeptide PN-A5 attenuates spontaneouspain in CIBP acutely.

FIG. 6B is a graph showing the results of tactile allodynia test usingvon Frey filaments.

FIGS. 7A-7F is a series of graphs showing the pain responses (guardingand flinching) of mice having a bone intramedullary transplantation of abreast cancer cell line under various single-dose therapeutic treatmentregimens and the blockade by the Mas receptor antagonist A779.

FIGS. 8A-8D is a series of bar graphs showing the pain responses(guarding and flinching) of mice having a bone intramedullarytransplantation of a breast cancer cell line under various chronictherapeutic treatment regimens and the blockade by the Mas receptorantagonist A779.

FIGS. 9A-D is a series of graphs showing the pain responses of micehaving an intramedullary transplantation of a breast cancer cell lineunder various chronic treatment regimens using either Ang-(1-7) orantagonists of the AT1 or AT2 receptor.

FIGS. 10A-10B is a series of graphs showing the nesting behavior ofexperimental mice with or without bone cancer under various treatmentregimens.

FIG. 11A is a Western blot showing Mas receptor expression in dorsalroot ganglia of nociceptive fibers in experimental animals.

FIG. 11B is a bar graph showing the relative expression of the Masreceptor between the ipsilateral and contralateral dorsal root gangliain experimental animals.

FIG. 11C is a Western blot showing Mas receptor expression in femoralexudate of experimental animals.

FIG. 11D is a bar graph showing the relative expression of the Masreceptor between the ipsilateral and contralateral femoral exudate inexperimental animals.

FIG. 12A is a series of photomicrograph of experimental animal femursfollowing various treatment regimens.

FIG. 12B is a bar graph showing the quantification of tumor tissue inthe experimental animal femurs.

FIG. 13A is a series of radiographs taken from experimental animals.

FIG. 13B is a graph quantifying bone lesion scoring in experimentalanimals.

FIG. 13C is a bar graph showing the amount of carboxy-terminal collagencrosslinks as a measure of bone integrity in experimental animals.

FIGS. 14A-C are a series of graphs showing the anti-nociceptive effectof PN-A5 in the acute/inflammatory model of carrageenan-induced pain.FIG. 14A is a line graph showing the raw threshold data from the vonFrey filament test. FIG. 14B is a line graph showing the normalized datafrom FIG. 14A. FIG. 14C is a bar graph showing the AUC calculated fromFIG. 14B.

FIG. 15A is model of the three-dimensional structure of native Ang(1-7).FIG. 15B is a computational model of various glycosylated Ang(1-7)derivatives.

FIG. 16 is a line graph showing the in vitro serum half-life of nativeAng(1-7) and various derivatives.

FIGS. 17A-B are a series of line graphs showing the serum (FIG. 17A) andCSF (FIG. 17B) concentration of native Ang(1-7) and PN-A5.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “native Ang-(1-7)” refers to the naturally-occurring Ang(1-7)polypeptide having the amino acid sequence Asp-Arg-Val-Tyr-Ile-His-Pro(SEQ ID NO: 2).

The term “Ang-(1-7) derivative” refers to oligopeptide in which one ormore amino acid residue is either modified or different than the aminoacid residue of the corresponding native Ang-(1-7). The term “Ang-(1-7)derivative” also includes oligopeptide of eight amino acid residues asdiscussed in more detail below.

By “PN-A2” is meant the Ang(1-7) derivative of SEQ ID NO: 3, which ishas the amino acid sequence of native Ang(1-7) except that Pro⁷comprises a C-terminal amidation (NH₂).

By “PN-A3” is meant the Ang(1-7) derivative of SEQ ID NO: 9, which ishas the amino acid sequence of native Ang(1-7) with the addition of aserine at the C-terminus (i.e., Ser⁸) and wherein Ser⁸ is glucosylatedand comprises a C-terminal amidation (NH₂).

By “PN-A4” is meant the Ang(1-7) derivative of SEQ ID NO: 9, which ishas the amino acid sequence of native Ang(1-7) with the addition of aserine at the C-terminus (i.e., Ser⁸) and wherein Ser⁸ is lactosylatedand comprises a C-terminal amidation (NH₂).

By “PN-A5” is meant the Ang(1-7) derivative of SEQ ID NO: 13, which ishas the amino acid sequence of native Ang(1-7) except that Pro⁷ issubstituted by Ser⁷ and wherein Ser⁷ is glucosylated and comprises aC-terminal amidation (NH₂).

By “PN-A6” is meant the Ang(1-7) derivative of SEQ ID NO: 13, which ishas the amino acid sequence of native Ang(1-7) except that Pro⁷ issubstituted by Ser⁷ and wherein Ser⁷ is lactosylated and comprises aC-terminal amidation (NH₂).

The term “carbohydrate” refers to pentose and hexose of empiricalformula (CH₂O)_(n), where n is 5 for pentose and 6 for hexose. Acarbohydrate can be monosaccharide, disaccharide, oligosaccharide (e.g.,3-20, typically 3-10, and often 3-5 monomeric saccharides are linkedtogether), or polysaccharide (e.g., greater than 20 monomeric saccharideunits). More often, the term carbohydrate refers to monosaccharideand/or disaccharide. However, it should be appreciated that the scope ofthe invention is not limited to mono- or di-saccharides. Often the terms“carbohydrate” and “saccharide” are used interchangeably herein.

The term “oligopeptide” as used throughout the specification and claimsis to be understood to include amino acid chain of any length, buttypically amino acid chain of about fifteen or less, often ten or less,still more often eight or less, and most often seven or eight.

It should be appreciated that one or more of the amino acids ofAng-(1-7) can be replaced with an “equivalent amino acid”, for example,L (leucine) can be replaced with isoleucine or other hydrophobicside-chain amino acid such as alanine, valine, methionine, etc., andamino acids with polar uncharged side chain can be replaced with otherpolar uncharged side chain amino acids. While Ang-(1-7) comprises 7amino acids, in some embodiments the oligopeptide of the invention haseight or less amino acids.

By “glycosylated,” is meant the covalent attachment to that amino acidof a mono-, di-, or polysaccharide. The glycosylation may be N-linked orO-linked, as appropriate. For example, N-linked glycosylation may occurat the R-group nitrogen in asparagine or arginine, and O-linkedglycosylation may occur through the R-group hydroxyl of serine,threonine, and tyrosine. Suitable carbohydrates include, for example,monosaccharides such as glucose, galactose, fructose, xylose, ribose,arabinose, lyxose, allose, altrose, mannose, fucose, and rhamnose,disaccharides such as sucrose, lactose, maltose, trehalose, melibiose,cellobiose, higher-order structures such as sorbitol, mannitol,maltodextrins, and farinose, and amino sugars such as galactosamine andglucosamine. In some particular embodiments, the polypeptide isglycosylated with glucose, lactose, cellobiose, melibiose, β-D-glucose,β-D-lactose, β-D-cellobiose, or β-D-melibiose.

The term “combinations thereof,” which reference to any modifications(e.g, carbohydrate modifications) of Ang-(1-7) derivatives refers tooligopeptides in which two, three, four, five, six, seven, or eight ofthe individual amino acids are modified by the attachment of acarbohydrate. For Ang-(1-7) derivatives having a plurality ofcarbohydrate modifications, the modifying carbohydrates may be the sameon every modified amino acid, or the several modified amino acids maycomprise a mixture of different carbohydrates.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal, at an appropriate interval and fora sufficient duration for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on the compound, the disease and its severity,physiological factors unique to the individual including, but notlimited to the age, weight, and body mass index, the unitary dosage,cumulative dosage, frequency, duration, and route of administrationselected.

“Prevent,” when used in connection with the occurrence of a disease,disorder, and/or condition, refers to reducing the risk of developingthe disease, disorder and/or condition for which the subject is at riskof developing

“Treat” refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, reduce severity of one or moresymptoms or features of a particular disease, disorder, and/or conditionin a subject diagnosed as having that disease or disorder.

The terms “approximately” or “about” in reference to a number aregenerally taken to include numbers that fall within a range of 5%, 10%,15%, or 20% in either direction (greater than or less than) of thenumber unless otherwise stated or otherwise evident from the context(except where such number would be less than 0% or exceed 100% of apossible value).

The term “subject” or “patient” refers to any organism to which acomposition of this invention may be administered, e.g., forexperimental, diagnostic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, dogs, cats,non-human primates, and humans).

By “dosing regimen” is meant a set of unit doses (e.g., one, two, three,four, or more) that is/are administered individually to a subject,typically separated by periods of time. In some embodiments, a dosingregimen comprises one or a plurality of doses each of which areseparated from one another by a time period. The time period separatingindividual doses may have a fixed or variable duration, or thetherapeutic agent may be administered on an as-need basis. A dosingregimen may span one day, multiple days, multiple weeks, multiplemonths, or be administered for the lifetime of the subject (e.g., 1, 2,3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or 1, 2, 3, 4, 5, 6, 9, or 12months or more). In some embodiments, the therapeutic agent isadministered once a day (QD), twice a day (BID), three times a day(TID), four times a day (QID), or less frequently (i.e., every second orthird day, one each week, or once each month).

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

Oligopeptides of the Invention:

The renin-angiotensin system (RAS), well known for roles in bloodpressure regulation and fluid homeostasis, was recently implicated inmetastatic bone disease including inflammation, angiogenesis, tumor cellproliferation, and migration. Angiotensin II (Ang II) is the major endproduct of the RAS through cleavage by Angiotensin Converting Enzyme(ACE). This nonapeptide binds to and activates two G-protein coupledreceptors (GPCRs): angiotensin II receptor type 1 (AT1) and type 2(AT2). Physiological effects such as vasoconstriction, inflammation,fibrosis, cellular growth/migration, and fluid retention are reportedfor AT1 and AT2. Ang II is cleaved by ACE2 to yield Angiotensin-(1-7)(Ang-(1-7)), a biologically active heptapeptide. In contrast to Ang II,Ang-(1-7) binds to the GPCR, Mas receptor (MasR; Kd=0.83 nM) with 60-100fold greater selectivity over the AT1 and AT2 receptors. Activation ofthe MasR elicits effects opposite to those of the Ang II/AT1/AT2 axisincluding having anti-inflammatory and antidepressant activities.

Some aspects of the invention provide oligopeptides that are derivativesof Ang-(1-7). As discussed above, the term “derivative” of Ang-(1-7)refers to an oligopeptide whose amino acid sequence of any one or moreof Ang-(1-7) is modified (e.g., via methylation, presence of afunctional group, such as hydroxy group on proline), attached to acarbohydrate, is replaced with corresponding D-amino acid or an“equivalent amino acid” as defined above, and/or the terminal aminogroup end or the carboxyl end of Ang-(1-7) is modified, for example, thecarboxylic acid end can be modified to be an amide, an amine, a thiol,or an alcohol functional group, or one in which an additional amino acidresidue is present compared to native Ang-(1-7). It should beappreciated that the term “Ang-(1-7) derivative” excludes the nativeAng-(1-7), i.e., amino acid sequences of endogenous Ang-(1-7) withoutany modification.

In some embodiments, oligopeptides of the invention have the amino groupon the carboxylic acid terminal end (i.e., the —OH group of thecarboxylic acid is replaced with —NR^(a)R^(b), where each of R^(a) andR^(b) is independently hydrogen or C₁-C₆ alkyl) and/or have one or moreamino acid residues that are (i) replaced with a corresponding D-aminoacid, (ii) glycosylated, (iii) replaced with another amino acid, (iv) ora combination thereof.

In one particular embodiment, the oligopeptide of the invention isAng-(1-7) derivative of the formula: A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ IDNO:1), where A¹ is selected from the group consisting of aspartic acid,glutamic acid, alanine, and a derivative thereof; A² is selected fromthe group consisting of arginine, histidine, lysine, and a derivativethereof; A³ is selected from the group consisting of valine, alanine,isoleucine, leucine, and a derivative thereof; A⁴ is selected from thegroup consisting of tyrosine, phenylalanine, tryptophan, and aderivative thereof; A⁵ is selected from the group consisting ofisoleucine, valine, alanine, leucine, and a derivative thereof; A⁶ isselected from the group consisting of histidine, arginine, lysine, and aderivative thereof; A⁷ is selected from the group consisting of proline,glycine, serine, and a derivative thereof; and A⁸ can be present orabsent, wherein when A⁸ is present, A⁸ is selected from the groupconsisting of serine, threonine, hydroxyproline, and a derivativethereof, provided (i) at least one of A¹-A⁸ is optionally substitutedwith a mono- or di-carbohydrate; or (ii) when A⁸ is absent: (a) at leastone of A¹-A⁷ is substituted with a mono- or di-carbohydrate, (b) A⁷ isterminated with an amino group, or (c) a combination thereof.

In some embodiments, A¹ is the amino terminal end of the oligopeptideand A⁸ (or A⁷ when A⁸ is absent) is the carboxyl terminal end. Still inother embodiments, A¹ is the carboxyl terminal end and A⁸ (or A⁷ when A⁸is absent) is the amino terminal end. Yet in other embodiments, thecarboxylic acid functional group of the carboxyl terminal end ismodified as an amide functional group, an amine functional group, ahydroxyl functional group, or a thiol functional group. The amide andthe amine functional groups can be non-alkylate, mono-alkylated ordi-alkylated.

Yet in other embodiments, the carbohydrate comprises glucose, galactose,xylose, fucose, rhamnose, or a combination thereof. In some instances,the carbohydrate is a mono-carbohydrate, whereas in other instances, thecarbohydrate is a di-carbohydrate.

In other embodiments, at least one of A¹-A⁸ is substituted with amono-carbohydrate. Still in other embodiments, at least one of A¹-A⁸ issubstituted with a di-carbohydrate. It should be appreciated that thescope of the invention also includes those oligopeptides having bothmono- and di-carbohydrates.

Exemplary di-carbohydrates that can be used in oligopeptides of theinvention include, but are not limited to, lactose, cellobiose,melibiose, and a combination thereof. However, it should be appreciatedthat the scope of the invention includes oligopeptides that aresubstituted with any dicarbohydrates known to one skilled in the art.

In one particular embodiment, A⁸ is serine or a derivative thereof. Insome instances, the carboxylic acid moiety of the serine is modified asan amide or an amine. In one case, serine is terminated as an aminogroup. Still in other embodiments, the serine residue of A⁸ isglycosylated with glucose or lactose.

Yet in other embodiments, at least one, typically at least two,generally at least three, often at least four, still more often at leastfive, yet still more often at least six, and most often all of A¹-A⁸ isD-amino acid.

Another aspect of the invention provides oligopeptides, such asAng-(1-7) derivatives, having eight amino acids or less, typically sevenor eight amino acid residues. In some embodiments, one or more aminoacids have attached thereto a carbohydrate group. Often the carbohydrategroup is attached to the oligopeptide via glycosylation. Thecarbohydrate can be attached to the oligopeptide via any of the sidechain functional group of the amino acid or the amide group.Accordingly, the scope of the invention includes, but is not limited to,O-glycosylate, N-glycosylate, S-glycosylated oligopeptides. The term“X-glycosylated” refers to having a carbohydrate attached to theoligopeptide via the heteroatom “X” of the amino acid. For example, forserine whose side-chain functional group is hydroxyl, “O-glycosylated”means the carbohydrate is attached to the serine's side-chain functionalgroup, i.e., the hydroxyl group. Similarly, “N-glycosylation” of leucinerefers to having the carbohydrate attached to the amino side-chainfunctional group of leucine. Typically, the glycosylation is on theside-chain functional group of the amino acid.

In some embodiments, the Ang-(1-7) derivative is glycosylated withxylose, fucose, rhamnose, glucose, lactose, cellobiose, melibiose, or acombination thereof.

Yet in other embodiments, the carboxylic acid terminal end of saidglycosylated Ang-(1-7) derivative is substituted with an amino group.When referring to the carboxyl acid terminal end being substituted withan amino group, it means —OH group of the carboxylic acid is replacedwith —NH₂ group. Thus, the actual terminal end functional group is anamide, i.e., rather than having the oligopeptide being terminated at thecarboxylic acid terminal end with a functional group —CO₂H, thecarboxylic acid terminal end is terminated with an amide group (i.e.,—CO₂NR′₂, where each R′ is independently hydrogen or C₁-C₁₂ alkyl).Still in other embodiments, the carboxylic acid terminal group isterminated with a hydroxyl or a thiol group. In some embodiments, themodified carboxylic acid terminal group is used to attach thecarbohydrate, e.g., via glycosylation.

One of the purposes of the invention was to produce Ang-(1-7)derivatives to enhance efficacy of action, in vivo stabilization, and/orpenetration of the blood-brain barrier. Improved penetration of theblood-brain barrier facilitates cerebral entry of the Ang-(1-7)derivative of the invention, and, consequently, Mas activation, orintrinsic-efficacy. To improve (i.e., increase) penetration of theblood-brain barrier, in some embodiments the Ang-(1-7) derivative isattached to at least one mono- or di-carbohydrates.

Without being bound by any theory, it is believed that the oligopeptideof the invention that are glycosylated exploits the inherentamphipathicity of the folded Ang-(1-7) glycopeptides (i.e., glycosylatedoligopeptides of the invention) and the “biousian approach” to deliverthe glycosylated oligopeptides of the invention across the blood-brainbarrier. In some instances, the amount of increase in crossing theblood-brain barrier by oligopeptides of the invention is at least 6%,typically at least 10%, and often at least 15% compared to nativeAng-(1-7). In some instances, the amount of increase in the Cmax foroligopeptides of the invention in cerebral-spinal fluid is 2-10 fold,3-8 fold, or 5-8 fold compared to native Ang-(1-7). In some instances,the amount of increase in the Cmax for oligopeptides of the invention incerebral-spinal fluid is 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold compared tonative Ang-(1-7). In other instances, oligopeptides of the inventionhave in vivo half-life of at least 20 min, at least 30 min, at least 40min, at least 50 min, at least 60 min, or at least 2, hours, at least 3hours, at least 4 hours, at least 5 hours or at least 6 hours. In someinstances, the amount of increase in the in vivo half-life foroligopeptides of the invention is 2-30 fold, 3-25 fold, 4-20 fold, 4-10fold, 10-25 fold, 15-25 fold, or 20-25 fold compared to nativeAng-(1-7). Alternatively, compared to native Ang-(1-7), oligopeptides ofthe invention exhibit at least 50 fold, typically at least 75 fold, andoften at least 100 fold increase in in vivo half-life.

In other embodiments, oligopeptides of the invention exhibit enhancedvascular efficacy. Without being bound by any theory, it is generallyrecognized that blood-brain barrier transport occurs via an absorptiveendocytosis process on the blood side of the endothelium of the braincapillaries followed by exocytosis on the brain side, leading to overalltranscytosis. It is also believed that for this process to be efficient,the oligopeptide must bind to the membrane for some period of time, andmust also be able to exist in the aqueous state for some period of time(biousian nature). Based on previous work from one of the presentinventors, it is believed that effective drug delivery and blood-brainbarrier transport requires a biousian glycopeptide that has at least twostates: (1) a state defined by one or more membrane-bound conformationsthat permit or promote endocytosis; and (2) a state defined by awater-soluble, or random coil state that permits “membrane hopping” and,presumably, vascular efficacy.

In general, the degree of glycosylation does not have a large effect onthe structure of the individual microstates. Thus, altering the degreeof glycosylation allows for the modulation of aqueous vs. membrane-boundstate population densities without significantly affecting the overallstructure of the oligopeptide. Moreover, it is believed thatglycosylation also promotes stability to peptidases, thereby increasingthe half-life of the Ang-(1-7) derivatives in vivo.

TABLE 1 sets forth some particularly useful Ang(1-7) derivativepolypeptides but is not intended to be limiting on the scope of theinvention. Amino Acid Position SEQ 1 2 3 4 5 6 7 8 ID NO: Asp Arg ValTyr Ile His Pro — 2 Asp Arg Val Tyr Ile His Pro° — 3 Asp Arg Val Tyr IleHis Pro* — 4 Asp Arg Val Tyr Ile His Pro°* — 5 Asp Arg Val Tyr Ile HisPro Ser 6 Asp Arg Val Tyr Ile His Pro Ser^(o) 7 Asp Arg Val Tyr Ile HisPro Ser* 8 Asp Arg Val Tyr Ile His Pro Ser°* 9 Asp Arg Val Tyr Ile HisSer — 10 Asp Arg Val Tyr Ile His Ser° — 11 Asp Arg Val Tyr Ile His Ser*— 12 Asp Arg Val Tyr Ile His Ser°* — 13 Ala Arg Val Tyr Ile His Pro — 14Ala Arg Val Tyr Ile His Pro° — 15 Ala Arg Val Tyr Ile His Pro* — 16 AlaArg Val Tyr Ile His Pro°* — 17 Ala Arg Val Tyr Ile His Pro Ser 18 AlaArg Val Tyr Ile His Pro Ser° 19 Ala Arg Val Tyr Ile His Pro Ser* 20 AlaArg Val Tyr Ile His Pro Ser°* 21 Ala Arg Val Tyr Ile His Ser — 22 AlaArg Val Tyr Ile His Ser^(o) — 23 Ala Arg Val Tyr Ile His Ser* — 24 AlaArg Val Tyr Ile His Ser°* — 25 Asp Arg Nle Tyr Ile His Pro — 26 Glu LysVal Ser Val Arg Ser Ala Ala Leu Thr Leu - or ° Cys Asn - or ° He Ala Nle-, °, *, Pro Ala - or ° Ala or °* Gly Gly Gly - or ° Lys Pro Tyr - or °Asp Arg Nle Tyr He His Pro Phe 27 Glu Lys Val Ser Val Arg Ala Ser AlaAla Leu Thr Leu - or ° Cys Asn - or ° Ile Ala Nle Ile Pro Ala - or ° AlaTyr Gly Gly Gly -, °, *, - or ° Lys or °* Pro Tyr - or ° ¹ - Where morethan one amino acid is indicated, the amino acids are presented in thealternative. — = unmodified °= glycosylated *= carboxy terminal NH₂

In some embodiments, only the C-terminal amino acid is glycosylated(i.e., Xaa⁸ or Xaa⁷ if Xaa⁸ is absent). In some embodiments, theAng(1-7) derivative polypeptide is glycosylated with glucose, lactose,cellobiose, melibiose, β-D-glucose, β-D-lactose, β-D-cellobiose, orβ-D-melibiose. In some embodiments, the polypeptide comprises anO-linked glycosylation (e.g., on the R-group of a serine). In someembodiments, the C-terminal serine is glycosylated.

In some embodiments, non-naturally-occurring amino acids and/or aminoacid substitutes (e.g., dicarboxylic acids) may be substituted for thenaturally-occurring amino acids in Ang(1-7) and any of the Ang(1-7)derivative polypeptides including, for example, in the Ang(1-7)derivative polypeptides of TABLE 1. For example, α,α-disubstituted aminoacids, N-alkyl amino acids, C-α-methyl amino acids, β-amino acids, andβ-methyl amino acids. Amino acids analogs useful in the presentinvention may include, but are not limited to, β-alanine, norvaline,norleucine, 4-aminobutyric acid, orithine, hydroxyproline, sarcosine,citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid,6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine,N-acetyl serine, N-formylmethionine, 3-methylhistidine and otherunconventional amino acids. For example,

Xaa¹ may be Acpc (1-aminocyclopentane carboxylic acid), Me2Gly(N,N-dimethylglycine), Bet (betaine,1-carboxy-N,N,N-trimethylmethanaminium hydroxide), Sar (sarcosine) orSuc (succinic acid);

Xaa² may be Cit (citrulline), Orn (ornithine), acetylated Ser, or Sar;

Xaa³ may be Nle (norleucine), hydroxyproline, Acpc, or Aib(2-aminoisobutyric acid);

Xaa⁴ may be Tyr(PO₃), homoserine, azaTyr (aza-α¹-homo-L-tyrosine);

Xaa⁵ may be Nle, hydroxyproline, Acpc, or Aib;

Xaa⁶ may be 6-NH₂-Phe(6-aminophenylalaine); and

Xaa⁸ may be Phe(Br) (p-bromo-phenylalanine; may be L- orD-phenylalanine).

In some embodiments, the Ang(1-7) derivative polypeptide does notcomprise the naturally-occurring amino acid sequence of native Ang(1-7)set forth in SEQ ID NO: 2.

In some embodiments, Ang(1-7) and any of the Ang(1-7) derivativepolypeptides, including those specifically defined in TABLE 1, maycomprise entirely L-amino acids, entirely D-amino acids, or a mixture ofL- and D-amino acids (e.g., having 1, 2, 3, 4, 5, 6, 7, or 8 D-aminoacids).

The Ang(1-7) and Ang(1-7) derivative polypeptides may be produced by anysuitable method including, without limitation, by peptide synthesismethods such exclusive solid phase synthesis, partial solid phasesynthesis, fragment condensation, classical solution synthesis,native-chemical ligation, and recombinant techniques.

Cognitive Dysfunction

Cognitive dysfunction or impairment is a common neurologicalcomplication of congestive heart failure (“CHF”) and post cardiacsurgery affecting approximately 50-70% of patients at hospital dischargeand 20-40% of patients six months after surgery. The occurrence of CHFand postoperative cognitive dysfunction is associated with increasedduration of hospitalization and impaired long-term quality of life.Without being bound by any theory, it is believed that in general anyclinical condition associated with an increase in inflammatory cytokinesand/or increase in reactive oxygen species in central nervous system, inparticular in the brain, can lead to cognitive dysfunction.

Other aspects of the invention provide methods for treating cognitivedysfunction and/or impairment in a patient using an oligopeptide of theinvention. Typically, methods of the invention include administering toa patient in need of such a treatment a therapeutically effective amountof an oligopeptide of the invention. It should be appreciated that theoligopeptides of the invention can be used to treat any clinicalconditions that are known to be treatable or appears to be treatableusing Ang-(1-7). However, for the sake of clarity and brevity, theinvention will now be described in reference to treating cognitivedysfunction and/or impairment in a patient.

The cognitive dysfunction that occurs in congestive heart failure (CHF)patients includes decreased attention, memory loss, psychomotor slowing,and diminished executive function, all of which compromises patients'ability to comply with complex medical regimens, adhere to dietaryrestrictions and make self-care decisions. Mechanisms thought tocontribute to cognitive impairment in patients with CHF include changesin cerebral blood flow, altered cerebrovascular autoregulation andmicroembolisms. In one study, cerebral blood flow was measured withsingle-photon emission computed tomography (SPECT) and found to bereduced by 30% in patients with severe heart failure. The causes fordecreased cerebral perfusion in CHF have been attributed to low cardiacoutput, low blood pressure and altered cerebrovascular reactivity. Insome cases, the cognitive impairment seen in CHF is improved followingeither heart transplant or improvement in cerebral blood flow viaoptimal management of CHF. However, for many patients with CHF,management is rarely optimal and the cognitive impairment persists.Interestingly, long-term follow up studies have revealed thatcognitively normal CHF patients have a significantly higher risk ofdementia or Alzheimer's disease compared to age-matched non-CHFcontrols, suggesting that CHF and cardiovascular disease predisposepatients to further cognitive impairment and dementia.

During CHF, the well characterized changes in the circulatingneurochemical milieu and increases in inflammatory factors are also seenin the brain. Most of the studies on CHF-induced changes in inflammatorycytokines and ROS have focused on brain regions involved in sympatheticoutflow regulation and not on cognition. CHF elevates sympathetic toneand causes abnormal cardiac and sympathetic reflex function. In the rat,ischemia-induced CHF significantly increases pro-inflammatory cytokinesand Angiotensin II type 1 receptors (AT1) in the paraventricular nucleus(PVN) of the hypothalamus. Further, in CHF rabbits, the increase insympathetic outflow is blocked by ICV injection of the super oxidedimustase (SOD) mimetic tempol, presumably by inhibition of ROS. CHF inthis model is associated with increased expression of NADPH oxidasesubunits and ROS production in the rostral ventral lateral medulla(RVLM) and increases in NO.

The role of ROS in learning and memory has been extensively studied. Allof the NAD(P)H oxidase subunits, including NOX2 and NOX4, have beenlocalized within the cell bodies and dendrites of neurons of the mousehippocampus and perirhinal cortex and are co-localized at synapticsites. These are key regions of the brain in learning and memory. In thebrain, superoxide production via actions of NAD(P)H oxidase are known tobe involved in neurotoxicity, age related dementia, stroke andneurodegenerative diseases and have been identified throughout the brainincluding the hippocampus, thalamus, cerebellum and amygdala. Inyounger, healthy animals ROS and NAD(P)H oxidase is shown to be requiredfor normal learning and hippocampal long-term potentiation (LTP). Recentstudies in mice lacking Mas have shown that Ang-(1-7) and Mas areessential for normal object recognition processing and blockade of Masin the hippocampus impairs object recognition. In addition, Ang-(1-7)facilitates LTP in CA1 cells and this effect is blocked by antagonism ofMas. In older animals or in CHF animals, an increase in ROS is linked toLTP and memory impairments.

Over the last decade, it has become recognized that renin angiotensinsystem (RAS) involves two separate enzymatic pathways providing aphysiological counterbalance of two related peptides acting at distinctreceptors. The well described ACE-AngII-AT1 receptor system is thoughtto be physiologically opposed and balanced by the ACE2-Ang-(1-7)-Massystem. Functionally, these two separate enzymatic pathways of RAS arethought to be involved in balancing ROS production and nitric oxide (NO)in the brain, microvasculature and peripheral tissues. Increases in AT1receptor activation are known to increase NAD(P)H oxidase and ROSgeneration which are both known to contribute to abnormal increases ofsympathetic nerve activity observed in CHF and hypertension. Thisincrease in AT1 receptor-induced ROS formation is thought to be opposedby ACE2-Ang-(1-7)-Mas inhibition of ROS formation. Ang-(1-7), themajority of which is produced from ACE2 cleavage of Ang II, decreasesROS production and increases NOS in the brain via activation Mas and,possibly through AT2 receptor.

Within the brain, the Mas receptor is known to be expressed on neurons,microglia and vascular endothelial cells. Further, all three of thesekey components that make up the “neurovascular unit” (neurons, microgliaand endothelial cells) are central players in neurogenic hypertensionand CHF-induced increases in brain inflammation and ROS production. BothCHF and hypertension increase circulating cytokines promoting ROSproduction within the “neurovascular unit”. The end-result of thisfeed-forward cascade is neuronal dysfunction and cognitive impairment.The ideal therapeutic candidate to treat cognitive impairment would bedesigned to interrupt this cascade by working at both sides of theblood-brain barrier, the brain vascular endothelium and neuronal cells.Ang-(1-7), acting at the Mas receptor, is known to have effects at bothendothelial cells and neurons. However, using a native Ang-(1-7) fortreating cognitive dysfunction and/or impairment is not suitable becausenative Ang-(1-7) is susceptible to enzymatic degradation. Moreover,native Ang-(1-7) does not readily cross the blood-brain barrier to besuitable as a therapeutic agent.

Without being bound by any theory, it is believed that one of theadvantages of using oligopeptides of the invention in treating cognitivedysfunction and/or impairment is that oligopeptides of the inventionhave enhanced endothelial “interaction” and brain penetration. It isbelieved that oligopeptides of the invention act at both endothelialcells and neurons thus inhibiting inter alia neurovascular ROSproduction and mitigating the brain inflammatory cascade.

Accordingly, oligopeptides the invention can be used to treat cognitiveimpairment and/or dysfunction (1) associated with pre- and/orpost-surgery dementia, or (2) observed in patients with congestive heartfailure, cardiovascular disease, or hypertension. More generally,oligopeptides of the invention are useful in treating cognitivedysfunction and/or impairment in a subject whose cognitive dysfunctionand/or impairment is clinically associated with an increase ininflammatory cytokines and/or increase in reactive oxygen species(“ROS”) in the central nervous system, in particular the brain. As usedherein, the term “clinically associated” refers to the root cause orunderlying cause of cognitive dysfunction and/or impairment (such as,but not limited to, memory loss) that when ameliorated results inreduction, prevention, treatment or reversal of cognitive dysfunctionand/or impairment. Exemplary clinical conditions associated with anincrease in inflammatory cytokines and/or increase in reactive oxygenspecies that can cause cognitive dysfunction and/or impairment include,but are not limited to, circulatory compromise, cardiovascular disease,hypertension, hypotension, congestive heart failure, stroke, embolism,surgery (e.g., postoperative recovery condition), dementia, Alzheimer'sdisease, disease related cognitive impairment, trauma related cognitiveimpairment, age-related dementia, postoperative related delirium and/orincrease in inflammatory cytokine and/or increase in reactive oxygenspecies within the central nervous system of said subject or acombination thereof.

Anti-Nociception and Analgesia

The inventions described herein are based, in part, on the discoverythat Mas receptor agonists, including the prototypical native Ang(1-7)polypeptide, induce analgesia. As described herein, the analgesicproperties of Ang-(1-7) and Ang(1-7) derivatives were evaluated inanimal models of nociception including cancer-induced bone pain (CIBP)and inflammatory pain. It is demonstrated that acute and chronicsystemic administration of Ang-(1-7) and/or Ang(1-7) derivativessignificantly reduced behavioral indicia of several types/modalities ofpain. Importantly, repeated administration of these Mas receptoragonists attenuated CIBP without loss of efficacy after 7 days. However,no significant change in nesting behaviors with or without treatmentswas observed, suggesting that the nesting is not representative ofpossible anxiety or depression in mice with CIBP.

To confirm that the effects of Ang(1-7) and Ang(1-7) derivatives aremediated by the Mas receptor, control experiments using the Mas receptorantagonist, A-779, were performed. The inhibition of guarding andflinching by Ang(1-7) were significantly prevented by administration ofA-779.

Repeated Ang-(1-7) administration did not significantly alter theexpression of MasR in the DRGs or femur extrudate demonstrating thatrepeated Ang-(1-7) dosing does not significantly alter MasR expressionin the DRGs containing soma of fibers innervating the bone-tumormicroenvironment. Consistent with these findings, analgesic tolerancewas not observed over the treatment paradigm.

It also was discovered that pre-administration of an AT1 receptorantagonist, Losartan potassium, further alleviates cancer-induced bonepain, yet by itself had no significant effect. It is hypothesized thatLosartan augments the effect to Ang-(1-7) in CIBP because AT1 antagonisminhibits Ang-(1-7) from acting similarly to Ang II at AT1, therebyallowing Ang-(1-7) to bind primarily to MasR to induce analgesia. TheAT2 antagonist, PD 123319, did not attenuate the effects of Ang-(1-7)nor result in enhanced pain relief, indicating that the AT2 receptordoes not play a role in CIBP. In addition, the Ang-(1-7) did notdemonstrate any changes in motor activity by measuring the amount oftime animals that received Ang-(1-7) remained walking on a slow rotatingrod.

In sum, these data demonstrate that Ang-(1-7) at the Mas receptor is forinhibiting pain in the tumor-nociceptor microenvironment. Ang-(1-7) didnot significantly change the tumor-induced degradation of the bone nordid it significantly alter tumor proliferation, further suggesting theanalgesic effect is directly towards inhibiting nociceptive activationand not due to changes in tumor burden. Mas receptor agonists such asAng(1-7) therefore induce primary analgesia through pharmacologicmechansims rather than secondarily through effects on the bone tissue oranti-neoplastic activity.

Methods Of Administration

Oligopeptides of the present invention can be administered to a patientto achieve a desired physiological effect. Preferably the patient is ananimal, more preferably a mammal, and most preferably a human. Theoligopeptide can be administered in a variety of forms adapted to thechosen route of administration, i.e., orally or parenterally. Parenteraladministration in this respect includes administration by the followingroutes: intravenous; intramuscular; subcutaneous; intraocular;intrasynovial; transepithelially including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal and nasal inhalation via insufflation and aerosol;intraperitoneal; and rectal systemic.

The active oligopeptide can be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it can beenclosed in hard or soft shell gelatin capsules, or it can be compressedinto tablets. For oral therapeutic administration, the activeoligopeptide may be incorporated with excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparation can contain at least 0.1% of active oligopeptide. Thepercentage of the compositions and preparation can, of course, be variedand can conveniently be between about 1 to about 10% of the weight ofthe unit. The amount of active oligopeptide in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.Preferred compositions or preparations according to the presentinvention are prepared such that an oral dosage unit form contains fromabout 1 to about 1000 mg of active oligopeptide.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin can be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier. Various other materials can be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules can be coated with shellac,sugar or both. A syrup or elixir can contain the active oligopeptide,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active oligopeptide can be incorporated intosustained-release preparations and formulation.

The active oligopeptide can also be administered parenterally. Solutionsof the active oligopeptide can be prepared in water suitably mixed witha surfactant such as hydroxypropylcellulose. Dispersion can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacterial and fungi. Thecarrier can be a solvent of dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars or sodium chloride. Prolonged absorption of theinjectable compositions of agents delaying absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activeoligopeptide in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

The therapeutic oligopeptides of the present invention can beadministered to a mammal alone or in combination with pharmaceuticallyacceptable carriers, as noted above, the proportion of which isdetermined by the solubility and chemical nature of the oligopeptide,chosen route of administration and standard pharmaceutical practice.

The physician will determine the dosage of the present therapeuticagents which will be most suitable for prophylaxis or treatment and itwill vary with the form of administration and the particularoligopeptide chosen, and also, it will vary with the particular patientunder treatment. The physician will generally wish to initiate treatmentwith small dosages by small increments until the optimum effect underthe circumstances is reached. The therapeutic dosage can generally befrom about 0.1 to about 1000 mg/day, and preferably from about 10 toabout 100 mg/day, or from about 0.1 to about 50 mg/Kg of body weight perday and preferably from about 0.1 to about 20 mg/Kg of body weight perday and can be administered in several different dosage units. Higherdosages, on the order of about 2× to about 4×, may be required for oraladministration.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES Example 1: Ang-(1-7) Derivative High-Throughput Screening (HTS)

For HTS, a sensitive and direct measure of nitric oxide (NO) productionin 2 separate cell lines is utilized, primary CA1 hippocampal neuronsand human umbilical vein endothelial cells (HUVEC). The use of primaryCA1 cells is self-evident for the study of central effects. In addition,the contribution of endothelial dysfunction to the progression of CHFand to the induction of cognitive impairment is clinically appreciated.The emerging picture that the Ang-(1-7) singling axis holds promise as atherapeutic target for endothelial dysfunction strongly indicates thatreversal of CHF-induced endothelial dysfunction as mechanism cannot beruled out. HUVEC are isolated from the human umbilical vein andcryo-preserved after primary culture. HUVEC is included as a second linefor the primary screen because these cells are the model in vitro systemfor the study of endothelial cell function and can be used to directlymeasure Mas-dependent NO production.

Cell culture. To isolate primary hippocampal CA1 neuronal cells, wholebrain was removed from neonatal rat pups (1-2 day old) and the corticesdissected away. The hippocampus was isolated and the CA1 field wasexcised and placed in buffer. After gentle disruption in digestionbuffer, the cells were counted, placed in culture media, and plated in a96-well format coated with poly-d-lysine. At the time of plating, cellswere approximately at 50% density and were allowed to culture to 70-80%density before starting the assay. Commercially available HUVEC (LifeTechnologies/Thermo Fisher) was thawed and plated (5000-10,000cells/well) in a 96-well format coated with gelatin. HUVEC cells wereallowed to culture overnight before starting the assay.

Cell Activation: The xCELLigence system Real-Time Cell Analyzer (RTCA),developed by Roche Applied Science, uses microelectronic biosensortechnology to do dynamic, real-time, label-free, and non-invasiveanalysis of cellular events including G-protein receptor activation ofcells. The RTCA analysis was utilized to measure the potency andrelative ability of oligopeptides of the invention and native Ang-(1-7)to activate human umbilical vascular endothelial cells (HUVEC) inculture. Following uniform cellular adherence based on a linear increasein cell impedance (CI), HUVECs were treated with Ang-(1-7) andoligopeptides of the invention. Each trace of the CI over time in FIG. 1represents the average of 4 wells normalized to CI at the time ofcompound addition. FIG. 1 shows the results from data acquired using thexCELLigence RTCA to measure the relative potency of PN-A3, PN-A4, PN-A5and native Ang-(1-7). A 100 nM administration of PN-A3, PN-A4 and PN-A5and 10 nM of PN-A3 and PN-A5 resulted in a significant (˜2-fold)increase in CI over the native Ang-(1-7) demonstrating that theoligopeptides of the invention have greater potency for cell activationthan native Ang-(1-7).

NO production assay. As a screen for mechanisms of action ofoligopeptides of the invention, the ability to increase NO production ofthree oligopeptides of the invention (PN-A3, PN-A4 and PN-A5) werecharacterized and compared to native Ang-(1-7). Human umbilical vascularendothelial cells (HUVEC) culture plates received fluorescence reactionbuffer (0.2 M phosphate buffer, pH 7, 1 mM EDTA, 0.1% glucose)containing diaminofluorescein-FM diacetate (DAF-FM, 1 μM) to measurereal-time NO production. Time-resolved (10 minutes) fluorescentintensity was detected using a BioTek Synergy 2 microplate reader withexcitation at 485 nm and emission at 535 nm. DAF-FM is a sensitiveflourometric derivative for the selective detection of NO in live cells.

FIG. 2 shows relative peak fluorescence intensity following 5 minutesexposure to native Ang-(1-7) and three oligopeptides of the invention.Values were normalized to control fluorescence. As expected, nativeAng-(1-7) induced a significant elevation of NO over control levels.More importantly, as shown in FIG. 2 , oligopeptides of the invention(namely PN-A3, PN-A4 and PN-A5) also elicited a significant elevation ofNO over control levels, with PN-A5 significantly enhancing NO productionover that seen with native Ang-(1-7), *=p<0.05. These resultsdemonstrate that oligopeptides of the invention increase NO productionsimilar to or greater than that of native Ang-(1-7).

FIG. 3A illustrates the ability of the select Mas receptor antagonists,A779, (C₃₉H₆₀N₁₂O₁₁) which is known to block native Ang-(1-7) NOproduction, to also block NO production induced by the oligopeptide ofthe invention, namely PN-A5. In these studies, HUVEC cells wereincubated with DAF-FM, 1 μM to measure real-time NO production. Cellswere treated with either PN-A5 alone (1.0 mM, n=10), PN-A5+A779 (n=6).Measurements were obtained using an Olympus 550 Confocal Microscope andanalyzed using Image J. Images were obtained every 10 sec. These resultsindicate that the oligopeptide PN-A5 actions are due to activation ofthe Mas receptor.

FIG. 3B shows the averaged effect of the select Mas receptorantagonists, A779, which is known to block native Ang-(1-7) NOproduction, to also block NO production induced by the oligopeptide ofthe invention, PN-A5. In these studies, HUVEC cells were incubated withDAF-FM, 1 μM to measure real-time NO production. Cells were treated witheither PN-A5 alone (1.0 mM, n=10), PN-A5+A779 (n=6), or the NO donorS-nitroso-N-acetylpenicillamine (SNAP). Fluorescent measurements wereobtained using an Olympus 550 Confocal Microscope and analyzed usingImage J. Images were obtained every 10 sec. The NO response produced byPN-A5 was completely blocked by A779 demonstrating that PN-A5's abilityto increase NO is due to PN-A5 actions on the Mas receptor.

Example 2: Effects of Ang-(1-7) Derivatives on Heart Failure (HF)Induced Cognitive Impairment:

A total of 33, male C57Bl/6J adult mice (Harlan, 8-10 weeks old) wereused. Mice were randomly assigned to either the sham (n=12) orcongestive heart failure (CHF) group (n=21). Experimental groups aredescribed as follows: sham+saline, CHF+saline, CHF+PN-A5. All mice priorto surgery were weighed and anesthetized. For the CHF mice, MI wasinduced by ligation of the left coronary artery (LCA). Under anesthesia(2.5% isoflurane in a mixture of air and O₂) a thoracotomy was performedat the fourth left intercostal space and the LCA permanently ligated toinduce a myocardial infarction (MI). Occlusion of the LCA was confirmedby observing blanching, a slight change in color of the anterior wall ofthe left ventricle downstream of the ligature. Sham mice underwent thesame procedure with the exception of ligating the LCA.

Following 8 weeks post MI surgery, CHF mice were treated with eitherdaily subcutaneous injections of the Ang-(1-7) derivative PN-A5 (1mg/kg/day) for 28 days or saline. After 21 days, animals were tested forobject recognition using a standard NOR test as described below. Afterapproximately 25 days of treatment, animals were tested for spatialmemory using the standard Morris water task as described below.

Novel Object Recognition (NOR): The apparatus consisted of an evenlyilluminated Plexiglas box (12 cm×12 cm×12 cm) placed on a table insidean isolated observation room. All walls of the apparatus were covered inblack plastic, and the floor was grey with a grid that was used toensure that the location of objects did not change between objectfamiliarization and test phases. The mouse behavior and exploration ofobjects was recorded with a digital camera. The digital image from thecamera was fed into a computer in the adjacent room. Two digitalstopwatches were used to track the time the mouse spent interacting withthe objects of the test. All data was downloaded to Excel files foranalysis. Triplicate sets of distinctly different objects were used forthe test.

The novel object recognition task included 3 phases: habituation phase,familiarization phase, and test phase. For the habituation phase, on thefirst and second day, mice were brought to the observation roomhabituated to the empty box for 10 min per day. On the third day, eachmouse had a “familiarization” trial with two identical objects followedby a predetermined delay period and then a “test” trial in which oneobject was identical to the one in the familiarization phase, and theother was novel. All stimuli were available in triplicate copies of eachother so that no object needed to be presented twice. Objects were madeof glass, plastic or wood that varied in shape, color, and size.Therefore, different sets of objects were texturally and visuallyunique. Each mouse was placed into the box the same way for each phase,facing the center of the wall opposite to the objects. To preclude theexistence of olfactory cues, the entire box and objects were alwaysthoroughly cleaned with 70% ethanol after each trial and between mice.During the familiarization phase, mice were allowed to explore the twoidentical objects for 4 min and then returned to their home cages. Aftera 2 hour delay, the “test phase” commenced. The mice were placed back tothe same box, where one of the two identical objects presented in thefamiliarization phase was switched to a novel one and the mouse wasallowed to explore these objects for another 4 min. Mouse “exploratorybehavior” was defined as the animal directing its nose toward the objectat a distance of ˜2 cm or less. Any other behavior, such as restingagainst the object, or rearing on the object was not considered to beexploration. Exploration was scored by an observer blind to the mouse'ssurgical group (CHF vs. Sham). Finally, the positions of the objects inthe test phases, and the objects used as novel or familiar, werecounterbalanced between the 2 groups of mice.

Discrimination ratios were calculated from the time spent exploring thenovel object minus time spent exploring the familiar object during thetest phase divided by the total exploration time. DRatio=(t novel−tfamiliar)/(t novel+t familiar). Data were analyzed from first 2 minutesof ‘test phase’. A positive score indicates more time spent with thenovel object, a negative score indicates more time spent with thefamiliar object, and a zero score indicates a null preference. All NORdata was examined using one-way analysis of variance, between subjects(ANOVA). Individual group differences were tested using the post hocTukey HSD test. In comparisons between groups of different sample sizes,equal variance was tested using a modified Levene's test. Allstatistical tests and p-values were calculated using MS Excel withDaniel's XLtoolbox and alpha was set at the 0.05 level. Error barsrepresent SEM.

Morris Water Task: Testing Spatial Learning and Memory/Visual Test: Theapparatus used was a large circular pool approximately 1.5 meters indiameter, containing water at 25° C. made opaque with addition ofnon-toxic white Crayola paint. An escape platform was hidden just belowthe surface of the water. Visual, high contrast cues were placed on thewalls of the test room. A digital camera connected to a computer in theadjacent room is suspended over the tank to record task progress. Forspatial testing prior to MI at 4 and 8 weeks post-MI or sham surgery,the platform was located at different sites in the pool.

During the spatial version of the Morris water task, all animals weregiven 6 training trials per day over 4 consecutive days. During thesetrials, an escape platform was hidden below the surface of water. Micewere released from seven different start locations around the perimeterof the tank, and each animal performed two successive trials before thenext mouse was tested. The order of the release locations waspseudo-randomized for each mouse such that no mouse was released fromthe same location on two consecutive trials. Performance on the swimtask was analyzed with a commercial software application (ANY-maze, WoodDale, Ill.). Because different release locations and differences inswimming velocity produce variability in the latency to reach the escapeplatform, a corrected integrated path length (CIPL) was calculated toensure comparability of mice performance across different releaselocations. The CIPL value measures the cumulative distance over timefrom the escape platform corrected by an animal's swimming velocity, andis equivalent to the cumulative search error. Therefore, regardless ofthe release location, if the mouse mostly swims towards the escapeplatform the CIPL value will be low. In contrast, the more time a mousespends swimming in directions away from the platform, the higher theCIPL value.

Following approximately 21 days of treatment with oligopeptide PN-A5,CHF mice showed object recognition memory improvement. FIG. 4illustrates the effects of three weeks treatment with oligopeptide PN-A5on object recognition memory as determined by the Novel ObjectRecognition Test (NOR). The mean performance of CHF mice witholigopeptide PN-A5 treatment (n=11) was similar to sham mice with saline(n=6), (CHF-Ang-(1-7) derivative PN-A5 M=+0.38, SE 0.11 vs. Sham-salineM=+0.52, SE 0.06) and significantly greater in comparison to CHF micetreated with saline (n=10) (M=−0.05, SE 0.09, *=p=0.009. These resultsdemonstrate that oligopeptide PN-A5 acts to attenuate and even rescueobject recognition memory impairment in mice with CHF.

Following approximately 25 days of treatment with oligopeptide PN-A5,CHF mice showed spatial memory improvement. FIG. 5 shows the mean CIPLof CHF+oligopeptide PN-A5 mice (n=11), CHF-saline treated mice (n=10)and Sham+saline mice (n=6). The CHF+oligopeptide PN-A5 mice showedsignificant improvement in spatial memory day 3 of the Morris swim taskas compared to CHF-saline mice. CHF mice treated with saline had asignificantly higher CIPL score as compared to CHF-oligopeptide PN-A5treated mice (CHF-saline M=32.5, SE=2.1 vs CHF-oligopeptide PN-A5M=23.5, SE 2.2, *=p=0.003. These results demonstrate that oligopeptidePN-A5 improves spatial memory.

Example 3: Effect of Ang(1-7) Derivatives on Cancer-Induced Bone Pain

BALB/cfC3H mice (Harlan, Ind., USA) were 15 to 20 g prior to initiationof study (n=5 animals per treatment group). Clinical signs of morbiditywere monitored and mice not meeting inclusion parameters (e.g.paralysis, rapid weight loss of >20% in 1 week) were removed from thestudy.

Mice were anesthetized with ketamine:xylazine (80 mg:12 mg/kg, 10 ml/kginjection volume; Sigma-Aldrich). An arthrotomy was performed. Thecondyles of the right distal femoris were exposed and a hole was drilledto create a space for injection of 4×10⁴ 66.1 cells in 5 μL Opti-MEM or5 μL Opti-MEM without cells in control animals within the intramedullaryspace of the mouse femoris. Injections were made with an injectioncannula affixed via plastic tubing to a 10-μL Hamilton syringe (CI31,Plastics One). Proper placement of the injector was confirmed throughuse of Faxitron X-ray imaging. Holes were sealed with bone cement.

Spontaneous pain (flinching and guarding), and tactile allodynia weremeasured 0, 15, 30, 60, 90 and 120 minutes after a single dose of drugwas administrated in a blinded fashion on Day 7. Breast cancer—inducedhypersensitivity returned to baseline levels 2 hours after drugadministration. Flinching and guarding were observed for duration of 2minutes during a resting state. Flinching was characterized by thelifting and rapid flexing of the right hind paw when not associated withwalking or movement. Flinches were recorded on a five-channel counter.Guarding was characterized by the lifting the right hind limb into afully retracted position under the torso. Time spent guarding over theduration of 2 minutes was recorded.

The assessment of tactile allodynia consisted of measuring thewithdrawal threshold of the paw ipsilateral to the site of tumorinoculation in response to probing with a series of calibrated von Freyfilaments using the Chaplan up-down method with the experimenter blindedto treatment groups. The 50% paw withdrawal threshold was determined bythe nonparametric method of Dixon.

On day 7, mice received an intraperitoneal (i.p.) injection of eithersaline or 0.8 μg/μL (200 μL) for a total dose of 800 μg/kg. The in-vivoefficacy of PN-A5 was measured for a total of 2 hours.

Within group data were analyzed by non-parametric one-way analysis ofvariance. Differences were considered to be significant if P≤0.05. Alldata were plotted in GraphPad Prism 6.

FIG. 6 shows the results on the effects of oligopeptide PN-A5 on cancerinduced bone pain (CIBP). Cancer implanted into the distal femoralis ofmice induced a significant increase in the number of spontaneousflinches (FIG. 6A) and time spent guarding (FIG. 6B) after 7 days.Administration of a bolus of PN-A5 (800 μg/kg, i.p.) significantlyreversed cancer induces spontaneous pain for nearly one hour in duration(flinching: 60 min; guarding: 30 min; p<0.001). Similarly,cancer-induced tactile hypersensitivity was significantly attenuated 30minutes after injection (p<0.01). For all measurements, the time of peakeffect was 15-30 min. Behaviors returned to post-surgery values 90 minpost-injection. Media inoculated, sham control animals did were notstatistically different from pre-surgery baselines at any point duringtime-course.

Example 4: Effect of Ang(1-7) Derivatives on Cancer-Induced Bone Pain:

The effect on cancer-induced bone pain of the archetypical Mas receptoragonist, native Ang(1-7) was investigated. It was discovered that theaction of Ang-(1-7) at the Mas receptor inhibits pain via thetumor-nociceptor microenvironment and not the tumor-bone environmentbecause Ang-(1-7) induced analgesia but did not significantly change thetumor-induced degradation of the bone or otherwise reduce bone loss.Chronic treatment with Ang-(1-7) did not significantly alter tumorproliferation, further suggesting the analgesic effect is directlytowards inhibiting nociceptive activation and not due to changes intumor burden.

Cell Culture: A murine mammary adenocarcinoma cell line, 66.1, wascultured in Eagle's minimum essential medium with 10% fetal bovineserum, 100 IU-1 penicillin, and 100 μg mL-1 streptomycin (P/S). The 66.1cells were plated in T-75 tissue culture flasks, allowed to growexponentially in an incubator at 37° C. and 5% CO₂. The viability ofcells cultured with treatments described below was measured using theXTT assay (ATCC, Manassas, Va.).

Animals: Female BALB/cAnNHsd mice (Harlan, Ind., USA) between 15 and 20g were used in this study. Mice were housed in a climate control room ona 12-hour light/dark cycle and allowed food and water ad libitum.Animals were monitored on days 0, 7, 10, and 14 of the study forclinical signs of rapid weight loss and signs of distress.

Drug Treatment: Animals received Angiotensin-(1-7) (Tocris, Ellisville,Mo.), the MasR antagonist A-779 (Abcam, Cambridge, Mass.), the AT1antagonist Losartan potassium (Tocris Bioscience, Minneapolis, Minn.),or the AT2 antagonist PD 123319 ditrifluoroacetate (Tocris Bioscience,Minneapolis, Minn.) dissolved in 0.9% saline. All intraperitoneal (i.p.)injections were made at a volume of 10 mL/kg. Systemic doses as follows:Ang-(1-7)=0-100 μg/kg, A-779=0.19 μg/kg, Losartan potassium=0.4 mg/kg,PD 123319 ditrifluoroacetate=0.4 mg/kg. In antagonist studies, A-779,Losartan potassium, or PD 123319 ditrifluoroacetate was administered 30minutes prior to Ang-(1-7).

Tail Flick: A warm water (52° C.) tail flick test was used to determinethe effects of Ang-(1-7) on acute nociception. The distal third of thetails of naïve mice were submerged into the water bath. The withdrawlatency, defined as the time for the tail to be withdrawn from the waterbath, was recorded. A cutoff time of 10 seconds was enforced to preventtissue damage. Baseline latencies were recorded prior to drugadministration. Animals were dosed (i.p.) with Ang-(1-7) (0-100 μg/kg).Tail flick latencies were reassessed 15, 30, 60, 90, 120, 150, and 180minutes post-treatment.

Rotarod: A rotarod performance test was used to determine the motorand/or sedative effects of Ang-(1-7) (Rotamex 4/8, Columbus Instruments,Columbus, Ohio, USA). Three days prior to testing, naïve mice weresubjected to 5 trials in which they were able to acclimate to therotating rod (10 revolutions/min). On the day of testing, animals wereallowed one trial and then baselined. The amount of time the animalremained on the rod was recorded, with a cutoff time of 120 seconds toprevent exhaustion. Animals were dosed (i.p.) as previously describedand reevaluated 15, 30, 60, and 120 minutes post-administration.

Arthrotomy—Intramedullary implantation of 66.1 cells: To induce CIBP, anarthrotomy was performed. Briefly, animals were anesthetized with 80mg/kg ketamine—12 mg/kg xylazine (in a 10 mL/kg volume). The surgicalarea was shaved and cleaned with 70% ethanol and betadine. The condylesof the right femur were exposed, and a burr-hole (0.66 mm) was drilledto create a space for the 66.1 cell inoculation. A 5 μl volume of 66.1cells (8,000 cells per 1 μl) in MEM (or 5 μl MEM without cells in shamanimals) was injected into the intramedullary space of the mouse femora.Proper placement of the injector was confirmed by radiograph (FaxitronX-ray imaging). Holes were sealed with bone cement and the patellareset. Muscle and skin were closed in separate layers with 5-0 vicrylsuture and wound autoclips, respectively. Animals were given 8 mg/kg (10mL/kg volume) gentamicin to prevent infection. Staples were removed 7days post-surgery.

Acute Behavioral Testing: Fourteen days post-surgery, baseline behaviorsof spontaneous flinching/guarding were recorded. Flinching wascharacterized by the lifting and rapid flexing of the hind pawipsilateral to femoral inoculation when not associated with walking orother movement. Guarding was characterized by the lifting the inoculatedhind limb into a fully retracted position under the torso. The totalnumber of flinches and the time spent guarding 2 min duration wasrecorded. Mice were then separated into treatment groups and dosedsystemically with Ang-(1-7) (0-10 μg/kg), A-779 (0.19 μg/kg), Losartanpotassium (0.4 mg/kg), PD 123319 (0.4 mg/kg), vehicle (0.9% saline), ora combination of Ang(1-7) and each antagonist. Antagonists wereadministered 30 minutes prior to Ang-(1-7). Following administration,animals were tested at over a three-hour time course until their painbehaviors returned to baseline

Chronic Behavioral Testing: Seven days post-surgery, baseline behaviorsof spontaneous pain, as described above, were recorded. Mice weretreated (i.p.) with Ang-(1-7) (0.058 μg/kg), A-779 (0.19 μg/kg), vehicle(0.9% saline), or a combination. Antagonist was administered 30 minutesprior to Ang-(1-7). Animals were dosed at the same time each day 7 to 14days post-surgery. On day 10, pain behaviors were assessed 15 minutesfollowing treatment, based on the time of peak effect determined by theacute studies. Fourteen days post-surgery, behaviors were again recordedpre- and post-treatment. Animals were sacrificed following treatment andtesting on the fourteenth day post-surgery, and the following tissueswere collected for biochemical analyses: serum, femur extrudate, andlumbar dorsal root ganglia.

Nesting: Nesting behaviors of naïve, media, and cancer-inoculated micewere assessed using the protocol described by Negus et al. Animals wereacclimated to individual cages, without an existing nest, for 30 minutesprior to drug administration. Cotton fiber nestlets were cut into 6equal pieces, and each piece was placed in the cage in 6 zones in themanner previously described following drug administration. Throughoutthe duration of the 100-minute time course, the number of cleared zoneswas recorded; upon completion the height (mm) of each fluffed nestletwas measured.

Western Blot Analysis: Dorsal root ganglia (DRG) and femur extrudatesfrom mice used in behavioral studies were analyzed for expression ofMasR. DRGs were homogenized in modified radioimmunoprecipitation assay(RIPA) buffer with protease inhibitor cocktail and EDTA (Pierce,Rockford, Ill., USA) via sonication. 10 μg of each sample was resolvedon a 10% SDS—polyacrylamide gels (TGX Criterion XT; Bio-Rad, Hercules,Calif.) and transferred to a polyvinylidene difluoride membrane (PVDF,Bio-Rad, Hercules, Calif.). Ipsilateral and contralateral femurs wereremoved from each animal. For each femur, the proximal and distal endswere clipped and the intramedullary extrudate was flushed six times with700 μL phosphate-buffered saline containing protease inhibitor cocktailand EDTA (Pierce, Rockford, Ill., USA). Femur marrow from five animalswas pooled per sample and 15 μg of sample was resolved and transferredin the same manner as DRGs. Protein transfer was verified by stainingblots with Ponceau S (Sigma, St. Louis, Mo.), and PVDF membranes wereblocked with 5% non-fat dry milk in Tris-buffered saline containing0.05% (v/v) Tween-20 (TBST) for one hour at room temperature. Membraneswere then incubated with primary antibody: rabbit polyclonalanti-Angiotensin-(1-7) Mas Receptor (Alomone Labs AAR-013; 1:200dilution for DRGs or 1:800 for femurs) or mouse monoclonal anti-actinAC40 (Cell Signaling 7076S; 1:4,000 dilution) in 1% milk in TBSTovernight at 4° C. The membranes were washed in TBST and incubated withappropriate secondary antibodies (Cell Signaling 7074 Anti-rabbit IgGHRP-Linked, 1:10,000 dilution; Cell Signaling 7076 Anti-mouse IgGHRP-Linked, 1:5000 dilution) for 1 hour at room temperature. Membraneswere again washed and developed using enhanced chemiluminescence(Clarity ECL Substrate, Bio-Rad, Hercules, Calif.), and bands weredetected using GeneMate Blue-Ultra Autorad films (BioExpress, Kaysville,Utah. Bands were quantitated and corrected for background using ImageJdensitometric software (Wayne Rasband, Research Services Branch,National Institute of Mental Health, Bethesda, Md.). All data werenormalized to actin in each lane and reported as fold change overuntreated control.

Ang-(1-7) Administration in Established CIBP Attenuates Spontaneous Painin a MasR Dependent Manner.

The antinociceptive efficacy of Ang-(1-7) in a model of established CIBPin which 66.1 tumor cells were injected into the right femurs ofsyngeneic BALB/cAnNHsd mice was investigated. Prior to surgery, mice didnot display behavioral signs of pain (data not shown). Animals showed asignificant amount of flinching (FIG. 7A at “post-Sx”) and guarding(FIG. 7B at “post-Sx”) compared to media-treated controls (p<0.0001,n=8). A single systemic injection of Ang-(1-7) (0.036, 0.360, 1, and 10μg/kg) or vehicle was administered, and pain behaviors were assessed.Animals given an acute i.p. administration of Ang-(1-7) showed asignificant (p<0.01, n=8) reduction in spontaneous pain behaviors withan onset 15 min after injection of either 0.36 or 1 μg/kg whichpersisted for nearly 2 hours (FIGS. 7A and 7C).

Dose response curves were constructed from data collected at the time ofpeak effect, 15 min, for guarding and flinching behavior (FIGS. 7B and7D, respectively). At 15 min, the maximum effect of Ang-(1-7) inreducing guarding behavior was 52.75% (p<0.01, n=8) with a correspondingA90 dose of 0.058 μg/kg (FIG. 7B). Flinching displayed less of adose-dependency and a more significant inhibition at the lower dose(0.036 μg/kg). Thus, a single injection of Ang-(1-7) is effective inreducing spontaneous pain behavior by more than 50% in animals withestablished CIBP.

To confirm that the observed Ang-(1-7) effect is mediated by the Masreceptor, A-779 (0.19 μg/kg), a selective MasR antagonist, or vehiclewas administered 30 minutes prior to Ang-(1-7) (0.058 μg/kg) 14 dayspost-femur inoculation. Inhibition of MasR with A-779 alone did notalter spontaneous or evoked pain thresholds; however, pretreatment withA-779 significantly inhibited Ang-(1-7) attenuation of guarding (FIG.7E, p<0.01) and flinching (FIG. 7F, p<0.001). These data demonstratethat Ang-(1-7) elicits antinociception in established CIBP throughactions at MasR.

Antinociceptive Effects of Ang-(1-7) through MasR are Maintained AfterRepeated Administration.

The antinociceptive activity of repeated Ang-(1-7) was investigated todetermine whether, like other analgesics, chronic administration resultstolerance. Ang-(1-7) (0.058 μg/kg, i.p.) was administered daily,beginning 7 days post implantation of 66.1 cells into the femur. Micewere evaluated for CIBP spontaneous pain behaviors on day 7 prior todrug administration, and on days 10 and 14 post-surgery 15 minutespost-treatment. Cancer inoculation significantly increased the amount oftime spent guarding and number of flinches 7 days post-surgery(p<0.0001, n=12). Animals experienced significant (p<0.0001, n=12)reduction in guarding (FIG. 8A) and flinching (FIG. 8B) followingAng-(1-7) treatment on days 10 and 14 post-surgery. Vehicle treatmenthad no significant effect.

A-779 was again used to confirm that the antinociceptive effects ofAng-(1-7) are mediated by the Mas receptor. A-779 (0.19 μg/kg) wasadministered 30 minutes prior to Ang-(1-7) (0.058 μg/kg) daily 7-14 dayspost-cancer inoculation (FIGS. 8C and 8D). Administration of A-779 alonehad neither a pro- or anti-nociceptive effect on the mice, and similarto earlier observations following a single injection, the chronicpre-treatment with A779 before Ang-(1-7) prevented attenuation of CIBPby the latter.

Effects of AT1/AT2 Antagonists on Ang-(1-7)/MasR-MediatedAntinociception in Established CIBP.

Ang II is the precursor molecule to Ang-(1-7). Accordingly, the role ofthe AngII receptors in mediating Ang-(1-7) antinociception wasinvestigated using a pre-treatment of the AT1 or AT2 selectiveantagonists, Losartan potassium (Ki=10 nM) and PD 123319 (also known asEMA200) (IC50=34 nM), respectively. Animals were inoculated with 66.1cells or media, as previously described, and pain behaviors wereassessed 14 days post-femur inoculation. Mice received either the AT1 orAT2 antagonist (0.4 mg/kg, i.p.) 30 minutes prior to Ang-(1-7) (0.058μg/kg, i.p.), or vehicle (0.9% saline). Spontaneous pain behaviors offlinching and guarding were recorded 15, 30, 60, 90, 120, and 150minutes post-administration. Confirming the previous results, Ang-(1-7)administration alone reduced pain behaviors (p<0.01, n=7), while neitherLosartan potassium nor PD 123319 significantly altered pain behaviorswhen administered alone. Interestingly, administration of the AT1receptor antagonist, Losartan potassium, prior to Ang-(1-7) yielded a77.527% maximal possible efficacy (MPE) in reducing guarding (p<0.0001,n=7) 30 minutes post-administration (FIG. 9A) and an 80.56% MPE inreducing flinching (p<0.0001, n=7) 15 minutes post-administration (FIG.9B). However, use of the AT2 antagonist, PD 123319, prior to Ang-(1-7)did not further increase nor decrease guarding or flinching of animalswith established CIBP (FIGS. 9C and 9D) as compared to the animal grouptreated with solely Ang-(1-7). The additive effect of Losartan potassiumto potentiate Ang-(1-7)-induced antinociception was not furtherinvestigated but may result from the ability of Losartan to preventAng-(1-7) binding to the AT1 receptor, thereby increasing the freeconcentration of Ang-(1-7) to bind to the Mas receptor.

Ang-(1-7) Administration in Established CIBP Does Not Change NestingBehaviors.

Nesting is an innate behavior in mice that is hindered by various statesof pain. The effect of both the arthrotomy and Ang-(1-7) administrationon nesting behavior was evaluated. Six equally sized pieces of nestletwere placed into 6 zones of the animals' individual cages. The number ofzones that the animals cleared of the nestlet pieces was recorded overthe 100-minute time course (FIG. 10A). During the first hour of thestudy on post-surgical day 6, the 66.1-inoculated animals clearedsignificantly fewer zones than the naïve animals (p<0.05). After thesecond hour of the study, the media animals cleared fewer zones thanboth the naïve and 66.1-inoculated animals (p<0.05). A second study wasconducted on post-surgical day 15 in which 66.1-inoculated animals weretreated with Ang-(1-7) (0.058 μg/kg, i.p.) or vehicle (0.9% saline)(FIG. 10B). The nesting behaviors of both treated cancerous groups didnot differ significantly from the naïve group. However, at both the 75and 90-minute time points, the media animals cleared fewer zones thanthe other groups (p<0.05). These data demonstrate that while the nestingof animals with established CIBP alters the nesting behaviors of mice,Ang-(1-7) administration does not further alter these complex behaviors.

Ang-(1-7) Administration Results in Antinociception But Not MotorImpairment in Naïve Mice

Ang-(1-7) was systemically administered (0.360, 1, 10 μg/kg, i.p.) tonaïve mice. Small but significant increase in thermal tail flicklatencies were observed (data not shown). Ang-(1-7) effects peakedbetween 15 and 30 min post administration with 1 μg/kg Ang-(1-7)(MPE=27.8%, p<0.001) and 10 μg/kg Ang-(1-7) (MPE=20.2%, p<0.01) thatreturned to baseline between 90 to 120 min.

To exclude the possibility that Ang-(1-7) administration reducedmobility in order to increase tail withdraw latency, rotarod testing wasperformed. Naïve animals were trained to walk on a rotating rod for 2min. After training, animals were injected with Ang-(1-7) by eitherspinal (0.3 pmol/5 μL) or systemic routes (0.058 and 10 μg/kg). Nosignificant differences in rotarod latencies were observed betweenvehicle and Ang-(1-7) treated mice (results not shown; p=0.99 i.t.;p=0.18 i.p.). Together, these data suggest that systemic Ang-(1-7) isantinociceptive after a single administration without noticeable impacton motility.

MasR is Expressed in the Dorsal Root Ganglion and Femur Extrudate.

Ipsilateral lumbar dorsal root ganglion (DRG) and femur extrudate werecollected from naive, sham, cancer (66.1), and 66.1 Ang-(1-7) treatedmice. In naïve mice, MasR is expressed in the dorsal root ganglia (FIG.11A); MasR bands were observed at ˜50 kDa and ˜40 kDa. Sham surgery(i.e. media only) or the introduction of the murine mammaryadenocarcinoma line 66.1 into the femoral intramedullary space did notsignificantly alter MasR expression levels in ipsilateral DRGs relativeto the contralateral control DRGs (FIG. 11B). MasR was also found to beexpressed in the femur extrudates of the same mice (FIG. 11C).Inoculation with the 66.1 cells significantly increased the expressionof MasR in the femur extrudate at both ˜50 kDa and ˜40 kDa (p<0.001compared to sham group), while sham surgery did not significantly alterthe expression of MasR in the femur extrudate as compared to the naïveanimal group (FIG. 11D).

Repeated Dosing of Ang-(1-7) Does Not Alter Tumor Burden of Mice withEstablished CIBP or Alter Cell Viability In Vitro.

Tumor burden was evaluated to determine whether the antinociceptiveeffect of repeated Ang-(1-7) administration results from anantineoplastic activity. Following chronic administration studies,femurs were harvested from animals, decalcified, and embedded inparaffin blocks prior to sectioning (5 micron) and hemotoxylin/eosinstaining (FIG. 12A). The region of the bone containing cancer cells wasquantified as a measure of total intramedullary content and representedas a percent of the entire cells within the bone. Repeated Ang-(1-7)administration did not significantly increase nor decrease the percenttumor of the bone (p=0.3, n=3-5) as compared to the saline-treated group(FIG. 12B). Thus, repeated Ang-(1-7) administration did notsignificantly impact tumor burden within the femur making it unlikelythat the antinociceptive effect is secondary to an antineoplasticactivity.

To verify in vivo findings, the effect of Ang-(1-7) on 66.1 cellviability was assessed in vitro. 66.1 cells were treated with vehicle,or increasing concentrations of Ang-(1-7) (1, 10, 100, or 1000 ng) for24 hr and an XTT cell viability assay was performed. As compared tovehicle treated cells (relative absorbance (RA±SD)=1.02±0.09), each ofthe four Ang-(1-7) treatments did not significantly change cellviability (1 ng: 0.93±0.11; 10 ng: 0.93±0.07; 100 ng: 0.78±0.11, 1000ng: 0.93±0.13). Together, these data indicate that Ang-(1-7) at thedoses/concentrations tested neither promotes tumor cell proliferationnor causes tumor cell death both in vivo and in vitro.

Repeated Dosing of Ang-(1-7) Does Not Affect Bone Remodeling of Micewith Established CIBP.

Radiographic images of all chronically treated animals were taken on day0, 7, 10, and 14 post-surgery to determine whether repeated Ang-(1-7)administration affected bone remodeling in mice with established CIBP(FIG. 13A). Day 14 images were scored by three blinded observers withthe following scale: 0—healthy bone, 1—1-3 lesions; 2—4-6 lesions;3—unicortical fracture; 4—bicortical fractures (FIG. 13B). A healthybone was defined as one without any visible lesions or fractures, and alesion was defined as a dark hole-like spot below the epiphyseal plate.While no animals experienced bicortical fractures, both saline andAng-(1-7)-treated cancer-inoculated animals experienced unicorticalfractures. Sham-treated (media) animals (7 out of 16) received scores of0. As another marker of bone remodeling, levels of carboxy-terminalcollagen crosslinks (CTX) in the serum were quantified (FIG. 13C). Whilecancer-inoculation significantly increased (p<0.05, n=3-4) CTX levels inthe bone compared to both media controls, Ang-(1-7) repeatedadministration did not significantly alter CTX levels compared to the66.1 saline treated group. Overall, daily Ang-(1-7) administration inmice with established CIBP did not significantly influence boneremodeling of the ipsilateral femur.

Example 5: Ang(1-7) Derivatives Mitigate Acute and Inflammatory Pain

The effect Mas receptor agonists was investigated in a model of acuteinflammatory pain. It was discovered that low doses of PN-A5 were moreeffective in reducing inflammation-induced allodynia than nativeAng(1-7) and a high dose of PN-A5.

Female BALB/cAnNHsd mice (Harlan, Ind., USA) between 15 and 20 g werefirst subjected to a the von Frey filament tactile allodynia test asdescribed above in order to establish a baseline response for eachanimal (“Pre-Car BL”). The right hindpaw of each test subject wasinjected subdermally with 50 μl carrageenan. Three hourspost-carrageenan, the subjects again were assessed using the von Freyfilaments in order to establish and untreated baseline response (“BL”)for each animal. Animals were immediately administered either 1, 3, or10 mg/kg PN-A5, 10 mg/kg native Ang(1-7), or saline vehicle byintraperitoneal injection (n=10). The von Frey filament test wasadministered to each animal 15, 30, 45, 60, 90, 120, and 180 minutesafter the carrageenan injection.

FIG. 14A provides the raw data for the von Frey response (threshold)showing that response to the filaments increases following carrageenaninjection. FIG. 14B shows the von Frey threshold data normalized usingpre-carrageenan baseline responses, expressed as % anti-allodynia,wherein 0% represents no therapeutic effect and 100% represents acomplete reversal of the carrageenan-induced allodynia. FIG. 14Cprovides a graphical representation of the area under the curve (AUC) ofthe data showing in FIG. 14B. These data together indicate that lowdoses of PN-A5 (1 mg/kg and 3 mg/kg) provide a substantial andstatistically significant reversal of carrageenan-induced allodynia,whereas a higher dose of PN-A5 and native Ang(1-7) (each administered at10 mg/kg) do not. Thus, PN-A5 alleviates pain and allodynia caused byinflammation.

Example 7: Glycosylation of Ang(1-7) and Its Derivatives ImprovesPharmacokinetic Properties

One known limitation of therapeutically administering native Ang(1-7) isits relatively short half-life and relatively poor blood-brain-barrierpermeability. The following experiments used a rational drug designapproach to assess the effect of adding various glycosides to Ang(1-7)and its derivatives on serum half-life and BBB permeability. Stabilityin vivo is affected by a number of factors, including susceptibility topeptidases and glycosidases, as well as aggregation phenomena insolution, and a wide array of binding events, including membraneabsorption. Interaction of the glycopeptide drug with biologicalmembranes is greatly influenced by both the geometry and degree ofglycosylation. Our previous experience with glycopeptide GPCR agonistsof a similar size indicates that the degree of glycosylation (mono-vsdisaccharide) will not greatly affect interaction with the MAS receptoror its activation.

Membrane-bound conformations of the Ang(1-7)-based glycopeptides weremodeled in silico by ¹H-NMR NOESY measurements in the presence ofd₂₅-SDS micelles. Using derived H—H distance constraints, a highlyamphipathic folded structure was characterized. As illustrated in FIG.15A, a Solvent Accessible Surface Area was constructed using the MOE®software package with the AMBER-99 force field to illustrate theresulting amphipathicity of the U-shaped folded structure. The unchargedlipophilic residues Val-Tyr-Ile are at the bottom of the “U” and insertinto the membrane while charged “ends” protrude into the aqueouscompartment. The “amphipathic moment” is suggested by the arrow.

FIG. 15B illustrates the MOE® calculations indicating that the linkagegeometries of the saccharide and peptide chain can modify interactionsof the resulting amphipathic glycopeptide with biological membranesprior to “docking” with the Mas receptor. D- or L-Serine, D- orL-Threonine, and D- or L-allo-Threonine, as well as D- or L-Cysteineorient the glycoside at different angles relative to the surface of themembrane.

Based on these calculations, native Ang(1-7), Ang(1-7) having aC-terminal amino group (Ang 1-7-NH₂; SEQ ID NO: 3; “PN-A2”), PN-A5 (Ang1-6-Ser(OGlc)-NH₂; SEQ ID NO: 13), and Ang 1-6-Ser(OLac)-NH₂ (Ang1-6-Ser(OLac)-NH₂; SEQ ID NO: 13) were produced and the serum half-lifetested. Serum half-life was assessed by incubating 100 μM of eachpeptide in mouse serum for eight hours. Aliquots were withdrawn at theindicated time intervals and the peptide concentration was determinedusing HPLC-MS and expressed as a percentage of the initialconcentration. As illustrated in FIG. 16 and Table 2, glycosylationsignificantly improved the serum half-life of the Ang(1-7) derivatives.

TABLE 2 In Vitro Serum Half-Life Assay Peptide Half-life Native Ang(1-7)14 min Ang 1-7-NH₂ (PN-A2) 21 min Ang 1-6-Ser(OGlc)-NH₂ (PN-A5) 1 hourAng 1-6-Ser(OLac)-NH₂ (PN-A6) 5.8 hours

Based on these findings, the in vivo serum stability and BBB penetrationwas assessed in vivo for Ang(1-7) and PN-A5. The peptides (10 mg/kg) orvehicle control were individually subcutaneously injected into naïvemice. Serum concentrations were determined every 10 minutes by HPLC-MSusing a 20-30 μl blood sample. Ang(1-7) and PN-A5 were found to reach amaximum serum concentrations of about 200 nM and about 3,500 nM,respectively (FIG. 17A). CSF samples were simultaneously withdrawn fromthe same animals via a microdialysis probe and assayed for the peptideconcentration and corrected for basal CSF levels. Ang(1-7) and PN-A5were found to reach a maximum CSF concentrations of about 50 nM andabout 400 nM, respectively (FIG. 17B).

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A method for treating a painful condition in asubject comprising administering a therapeutically effective amount ofan oligopeptide having the formula: A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸ (SEQ IDNO:1) wherein A¹ is selected from the group consisting of alanine andglutamic acid; A² is selected from the group consisting of arginine,histidine, and lysine; A³ is selected from the group consisting ofvaline, alanine, isoleucine, and leucine; A⁴ is selected from the groupconsisting of tyrosine, phenylalanine, and tryptophan; A⁵ is selectedfrom the group consisting of isoleucine, valine, alanine, and leucine;A⁶ is selected from the group consisting of histidine, arginine, andlysine; A⁷ is serine; and A⁸ is absent.